ObjectiveTo require hemagglutinin (HA) proteins for the study on the relationships between two kinds of amino acid alterations (H5 I151F, H5 I151F+A134V+E186D) and human receptor binding characteristics. MethodsVector pRB21’ was constructed, vp37-vaccinia virus vRB12 and pRB21’ were coinfected and transfected. Recombinant vaccinia viruses (re VV) were generated through homologous recombination, expressing HA proteins, which were identified with Western Blotting. ResultsThe vector pRB21’ within full length of H5 HA segments were obtained. Two kinds of HA proteins (re VV H5 I151F, re VV H5 I151F+A134V+E186D) were expressed by recombinant vaccinia viruses.ConclusionFor the first time recombinant vaccinia viruses were generated to express hemagglutinin of the avian influenza virus subtype H5N1, which pave the way for further studying the possibilities of human to human transmission caused by avian influenza viruses.